The enzyme, telomerase, appears active in nearly all tumors. Telomerase activity is a potential marker for the development of malignant tumors (Nelson, 1996). Telomerase activity is most frequently detected by the PCR based Telomeric Repeat Amplification Protocol (TRAP) developed by Kim et al., (1994). However, the original TRAP assay had several limitations. The assay is laborious and time-consuming. The original assay used radioisotope labeling techniques. Such techniques require special laboratory precautions. The assay is difficult to quantify due to the assay's non-linearity. The inhibition of Taq polymerase activity by cellular components in clinical specimens may lead to the false negative results. The assay requires special preparation and preservation of the specimens. Finally, the assay is difficult to automate. The TRAP assay has been modified to achieve better sensitivity, eliminate the hazardous radioactive labeling and represent results in a semi-quantitative mode (Wright et al., 1995, Ohyashiki et al., 1996).
The molecular structure of human telomerase is believed to be similar to the enzyme of other organisms. The enzyme derived from Tetrahymena is believed to be composed of a first protein subunit (p80), a second protein subunit (p95), and a telomerase RNA molecule. The two protein subunits are bound each other and at the same time manifest a specific affinity to nucleic acid sequences. The first subunit binds telomerase's RNA component with high affinity and specificity. The second subunit binds the telomeric DNA primer sites. As used herein, the term "first subunit" refers to such subunit which binds the RNA component, even though such subunit may have a molecular weight other than 80 kilodaltons. Similarly, the term "second subunit" refers to such subunit which binds the telomeric DNA, even though such subunit may have a molecular weight other than 95 kilodaltons. These two proteins and two nucleic acid components form an RNP complex. The RNP complex is stable and at the same time accesible for a synthesis of the telomere DNA in the region where such synthesis actually is occurring (Collins et al., 1995).
Human telomerase RNA, hTR, is composed of 445 nucleotides. The sequence is set forth in Seq. ID No 1 below:
5'-GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG GTGGCCAUUU Seq ID No. 1 - UUUGUCUAAC CCUAACUGAG AAGGGCGUAG GCGCCGUGCU UUUGCUCCCC - GCGCGCUGUU UUUCUCGCUG ACUUUCAGCG GGCGGAAAAG - CCUCGGCCUG CCGCCUUCCA CCGUUCAUUC UAGAGCAAAC AAAAAAUGUC - AGCUGCUGGC CCGUUCGCCU CCCGGGGACC UGCGGCGGGU CGCCUGCCCA - GCCCCCGAAC CCCGCCUGGA GCCGCGGUCG GCCCGGGGCU UCUCCGGAGG - CACCCACUGC CACCGCGAAG AGUUGGGCUC UGUCAGCCGC GGGUCUCUCG - GGGGCGAGGG CGAGGUUCAC CGUUUCAGGC CGCAGGAAGA - GGAACGGAGC GAGUCCCGCC GCGGCGCGAU UCCCUGAGCU GUGGGACGUG - - CACCCAGGA
(Feng et al., 1995, Zaug et al., 1996).
A need exists for improved diagnostic and analytical methods to detect the presence or absence of telomerase and to measure the levels of enzyme activity in clinical samples and specimens. Such an assay has utility in the diagnosis of various cancers and identification of metastasises. A need also exists to detect telomerase enzyme with the analytical methods of nucleic acid chemistry. Furthermore, a need exists to develop a telomerase detection assay that does not require expensive equipment, large laboratory facilities or highly trained technical personnel. Ideally, a test will be inexpensive, non-labor-intensive and capable of automation.